Composition for reducing inhibition of nucleic acid amplification

ABSTRACT

A composition for reducing the inhibitory effects of contaminants on nucleic acid amplification is provided. The composition includes effective amounts of ferric iron, an organic iron-chelating reagent, and a non-ionic surfactant. Optionally, the composition includes polyvinylpyrrolidone. The composition has a pH of about 8.45 to 8.85. The organic iron-chelating reagent has a first affinity constant greater than or equal to 104.2 with respect to ferric iron and a second affinity constant less than 103.8 with respect to magnesium. The first affinity constant and the second affinity constant are determined in deionized water at pH 8.45 and 20° C. Methods of using the composition to prepare a sample for nucleic acid amplification are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofPCT/US2015/066968, filed Dec. 21, 2015, which claims the benefit of bothU.S. Provisional Application No. 62/136,682, filed Mar. 23, 2015, andU.S. Provisional Application No. 62/096,217, filed Dec. 23, 2014, thedisclosures of which are incorporated by reference in their entiretyherein.

BACKGROUND

Conventional methods for the detection of pathogens and othermicroorganisms are based on culture methods, but these are timeconsuming, laborious, and no longer compatible with the needs of qualitycontrol and diagnostic laboratories to provide rapid results.

Efforts to overcome problems like culturing the microorganisms, falsepositives in pathogen detection have led to the development of genetictesting such as DNA-based diagnostic methods or nucleic acidproliferation methods. The use of DNA-based methods derives from thepremise that each species of pathogen carries unique DNA or RNAsignature that differentiates it from other organisms. These techniquesare the most promising and are increasingly used for rapid, sensitiveand specific detection of microbes.

Advances in biotechnology have led to the development of a diverse arrayof assays for efficient nucleic acid amplification.

The effective genetic testing of samples containingmicroorganisms/pathogens requires rapid sensitive assay methods thatgives instant or real time results. Time and sensitivity of analysis andinhibition of nucleic acid amplification caused by inhibitory substancesin the sample are certain limitations related to the usefulness ofgenetic testing.

It is desirable to have a composition and a method to efficiently andrapidly reduce or eliminate the inhibition of the nucleic acidamplification of the intended target.

SUMMARY

The present disclosure provides a composition for eliminating sampleinhibition in nucleic acid amplification reaction and a nucleic acidamplification method using this composition.

In a first aspect, an aqueous composition is provided for eliminatingsample inhibition in a nucleic acid amplification reaction. Thecomposition can comprise an organic iron-chelating reagent, ferric iron,and a non-ionic surfactant at a concentration greater than or equal to0.005% (mass/volume). The composition can have a pH of about 8.45 to8.85. The organic iron-chelating reagent can have a first affinityconstant greater than or equal to 10^(4.2) with respect to ferric ironand a second affinity constant less than 10^(3.8) with respect tomagnesium, wherein the first affinity constant and the second affinityconstant are determined in deionized water at pH 8.45 and 20° C.

In any of the above embodiments, the organic iron-chelating reagent cancomprise a plurality of carboxylate groups. In any of the aboveembodiments, the composition further can comprise2-hydroxypropane-1,2,3-tricarboxylate. In any of the above embodiments,the organic iron-chelating reagent can comprise EGTA, wherein a molarratio of the ferric iron to the EGTA is about 0.04 to about 0.28. In anyof the above embodiments, the composition further can comprisepolyvinylpyrrolidone.

In a second aspect, the present disclosure provides a nucleic acidamplification method, said method comprising a) contacting a compositioncomprising an organic iron-chelating reagent; ferric iron,polyvinylpyrrolidone, and a non-ionic surfactant with a target sample toform an aqueous mixture, wherein the aqueous mixture has a pH of about8.45 to 8.85, wherein the non-ionic surfactant is present in thecomposition at a concentration greater than or equal to 0.005%(mass/volume); b) subjecting the aqueous mixture of step a) to thermallysis; and c) subsequent to step b), subjecting the aqueous mixture toisothermal nucleic acid amplification. The organic iron-chelatingreagent has a first affinity constant greater than or equal to 10^(4.2)with respect to ferric iron and a second affinity constant less than10^(3.8) with respect to magnesium, wherein the first affinity constantand the second affinity constant are determined in deionized water at pH8.45 and 20° C.

In an embodiment, the present disclosure provides an isothermalamplification method, said method comprising a) contacting a compositioncomprising an organic iron-chelating reagent; ferric iron,polyvinylpyrrolidone, and a non-ionic surfactant with a target sample toform an aqueous mixture, wherein the aqueous mixture has a pH of about8.45 to 8.85, wherein the non-ionic surfactant is present in thecomposition at a concentration greater than or equal to 0.005%(mass/volume); b) subjecting the mixture of step a) to thermal lysis;and c) subsequent to step b), subjecting the mixture to isothermalnucleic acid amplification. The organic iron-chelating reagent has afirst affinity constant greater than 10^(4.2) with respect to ferriciron and a second affinity constant less than 10^(3.8) with respect tomagnesium, wherein the first affinity constant and the second affinityconstant are determined in deionized water at pH 8.45 and 20°.

In a third aspect, the present disclosure provides a kit comprisingferric iron, an organic iron-chelating reagent, and a non-ionicsurfactant. The organic iron-chelating reagent has a first affinityconstant greater than 10^(4.2) with respect to ferric iron and a secondaffinity constant less than 10^(3.8) with respect to magnesium, whereinthe first affinity constant and the second affinity constant aredetermined in deionized water at pH 8.45 and 20°.

In any embodiment of the kit, the organic iron-chelating reagent cancomprise a plurality of carboxylate groups. In any of the aboveembodiments, the kit further can comprise2-hydroxypropane-1,2,3-tricarboxylate. In any of the above embodimentsof the kit, the organic iron-chelating reagent can comprise ethyleneglycol tetraacetic acid, wherein a molar ratio of the ferric iron to theethylene glycol tetraacetic acid is about 0.04 to about 0.28. In any ofthe above embodiments, the kit further can comprise a component selectedfrom the group consisting of polyvinylpyrrolidone, a fluorosurfactant,an indicator dye, a preservative, a buffering agent, and an enhancer ofLAMP-BART reaction and combinations thereof.

The foregoing has outlined some pertinent objects of the disclosure.These objects should be construed to be merely illustrative of some ofthe more prominent features and applications of the intended disclosure.The disclosure includes other features and advantages which will bedescribed or will become apparent from the following more detaileddescription of the embodiment.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

Additional details of these and other embodiments are set forth in theaccompanying drawings and the description below. Other features, objectsand advantages will become apparent from the description and drawings,and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

The foregoing summary, as well as the following detailed description ofthe disclosure will be better understood when read in conjunction withthe appended drawings. For the purpose of assisting in the explanationof the disclosure, there are shown in the drawings embodiments which arepresently preferred and considered illustrative. It should beunderstood, however, that the disclosure is not limited to the precisearrangements and instrumentalities shown therein. In the drawings:

FIG. 1 shows a graphical representation of the effect of ferric ammoniumcitrate (FAC) in the LAMP-BART reaction with a sample containingprotein.

FIG. 2 shows a boxplot of Time to Peak (TTP) showing the effect of FACin the LAMP-BART reaction with a sample containing esculin or6,7,-dihydroxycoumarin.

FIG. 3 shows a boxplot of MC TTP showing the effect of surfactant vis avis FAC in the LAMP-BART reaction.

DETAILED DESCRIPTION

Before any embodiments of the present disclosure are explained indetail, it is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangement ofcomponents set forth in the following description or illustrated in thefollowing drawings. The invention is capable of other embodiments and ofbeing practiced or of being carried out in various ways. Also, it is tobe understood that the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting. Theuse of “including,” “comprising,” or “having” and variations thereofherein is meant to encompass the items listed thereafter and equivalentsthereof as well as additional items. It is to be understood that otherembodiments may be utilized and structural or logical changes may bemade without departing from the scope of the present disclosure. Thepresent disclosure will now be described more fully herein after. Forthe purposes of the following detailed description, it is to beunderstood that the disclosure may assume various alternative variationsand step sequences, except where expressly specified to the contrary.Thus, before describing the present disclosure in detail, it is to beunderstood that this disclosure is not limited to particularlyexemplified systems or embodiments that may of course, vary. The use ofexamples anywhere in this specification including examples of any termsdiscussed herein is illustrative only, and in no way limits the scopeand meaning of the disclosure or of any exemplified term. Likewise, thedisclosure is not limited to various embodiments given in thisspecification.

As used herein, the singular forms “a,” “an,” and “the” include pluralreference unless the context clearly dictates otherwise. The term“and/or” means one or all of the listed elements or a combination of anytwo or more of the listed elements.

When the term “about” is used in describing a value or an endpoint of arange, the disclosure should be understood to include both the specificvalue and end-point referred to.

As used herein the terms “comprises”, “comprising”, “includes”,“including”, “containing”, “characterized by”, “having” or any othervariation thereof, are intended to cover a non-exclusive inclusion.

As used herein, the phrase “nucleic acid,” and “nucleic acid sequence,”are interchangeable and not intended to be limiting. “Nucleic acid”shall have the meaning known in the art and refers to DNA (e.g., genomicDNA, cDNA, or plasmid DNA), RNA (e.g., mRNA, tRNA, or rRNA), and PNA. Itmay be in a wide variety of forms, including, without limitation,double-stranded or single-stranded configurations, circular form,plasmids, relatively short oligonucleotides, peptide nucleic acids alsocalled PNA's and the like. The nucleic acid may be genomic DNA, whichcan include an entire chromosome or a portion of a chromosome. The DNAmay include coding (e.g., for coding mRNA, tRNA, and/or rRNA) and/ornoncoding sequences (e.g., centromeres, telomeres, intergenic regions,introns, transposons, and/or microsatellite sequences). The nucleic acidmay include any of the naturally occurring nucleotides as well asartificial or chemically modified nucleotides, mutated nucleotides, etc.The nucleic acid can include a non-nucleic acid component, e.g.,peptides (as in PNA's), labels (radioactive isotopes or fluorescentmarkers), and the like.

As used herein, “amplifying” and “amplification” refers to a broad rangeof techniques for increasing polynucleotide sequences, either linearlyor exponentially. Exemplary amplification techniques include, but arenot limited to, polymerase chain reaction (PCR) or any other methodemploying a primer extension step. Other non-limiting examples ofamplification include, but are not limited to, ligase detection reaction(LDR) and ligase chain reaction (LCR). Amplification methods maycomprise thermal-cycling or may be performed isothermally such as Loopmediated isothermal amplification (LAMP-BART). In various embodiments,the term “amplification product” or “amplified product” includesproducts from any number of cycles of amplification reactions.

As used herein, the “polymerase chain reaction” or PCR is anamplification of nucleic acid consisting of an initial denaturation stepwhich separates the strands of a double stranded nucleic acid sample,followed by repetition of (i) an annealing step, which allowsamplification primers to anneal specifically to positions flanking atarget sequence; (ii) an extension step which extends the primers in a5′ to 3′ direction thereby forming an amplicon polynucleotidecomplementary to the target sequence, and (iii) a denaturation stepwhich causes the separation of the amplicon from the target sequence.Each of the above steps may be conducted at a different temperature,preferably using an automated thermocycler.

As used herein, “isothermally amplified” or “isothermal amplification”and like terms refers to a method of amplifying nucleic acid that isconducted at a constant temperature in contrast to amplifications thatrequire cycling between high and low temperatures unlike traditional PCRreactions. This requires that the DNA polymerase is a DNA polymerasehaving strand displacement activity. Isothermal amplifications are oftenconducted at substantially a single temperature because primers bind todisplaced DNA strands. In isothermal amplifications the reaction mixturecomprising the nucleic acid sample and optionally all primers may beheated to a denaturation temperature at which double-stranded nucleicacid in the reaction mixture denatures into single strands (e.g., atleast 85° C. to 90° C.) prior to the amplification and optionally priorto addition of the DNA polymerase when the DNA polymerase is inactivatedat the denaturation temperature.

As used herein, the terms “intended target”, “target nucleic acidregion,” “target specific nucleic acid,” “target region,” “targetsignature sequence” “target nucleic acid(s)”, “target nucleic acidsequences,” “target” or “target polynucleotide sequence” refers to anucleic acid of interest.

As used herein, “detecting” or “detection” refers to the disclosure orrevelation of the presence or absence in a sample of a targetpolynucleotide sequence or amplified target polynucleotide sequenceproduct. The detecting can be by end point, real-time, enzymatic, and byresolving the amplification product on a gel and determining whether theexpected amplification product is present, or other methods known to oneof skill in the art.

As used herein the term “sample” refers to a starting material suspectedof containing a nucleic acid. Detecting the nucleic acid in the sampleenables one to detect the presence of a microorganism. Examples ofsamples include, but are not limited to, food samples (including but notlimited to samples from food intended for human or animal consumptionsuch as processed foods, raw food material, produce (e.g., fruit andvegetables), legumes, meats (from livestock animals and/or gameanimals), fish, sea food, nuts, beverages, drinks, fermentation broths,and/or a selectively enriched food matrix comprising any of the abovelisted foods), water samples, environmental samples (e.g., soil samples,dirt samples, garbage samples, sewage samples, industrial effluentsamples, air samples, or water samples from a variety of water bodiessuch as lakes, rivers, ponds etc.), air samples (from the environment orfrom a room or a building), clinical samples, samples obtained fromhumans suspected of having a disease or condition, veterinary samples,forensic samples, agricultural samples, pharmaceutical samples,biopharmaceutical samples, samples from food processing andmanufacturing surfaces, and/or biological samples. Examples for nonfoodsamples as per the present disclosure may be culture broths. “Culturebroth” as used herein refers to a liquid medium for culturing themicroorganism.

As used herein, an “inhibitor” means any compound, substance, orcomposition, or combination thereof, that acts to decrease the activity,precision, or accuracy of an assay, either directly or indirectly, withrespect to the activity, precision, or accuracy of the assay when theinhibitor is absent. An inhibitor can be a molecule, an atom, or acombination of molecules or atoms without limitation.

In accordance to one aspect of the present disclosure, the term“inhibitors” as used herein refers to inhibitors of enzymes used inamplification reactions, for example. Examples of such inhibitorstypically include but not limited to proteins, peptides, lipids,carbohydrates, heme and its degradation products, urea, bile acids,humic acids, polysaccharides, cell membranes, and cytosolic components.The major inhibitors in human blood for PCR are hemoglobin, lactoferrin,and IgG, which are present in erythrocytes, leukocytes, and plasma,respectively. Examples of inhibitors also include iron ions or saltsthereof, other metal salts such as alkali metal ions, transition metalions etc., and indicator dyes present in growth medium.

In an embodiment of the present disclosure, esculin which is anindicator dye may act as inhibitor. Esculin is a coumarin glucoside(6-(beta-D-glucopyranosyloxy)-7-hydroxy-2H-1-benzopyran-2-one, CAS No.531-75-9) obtained from Aesculus hippocastanum (the horsechestnut) andis characterized by its fine blue fluorescent solutions. Esculin isgenerally added to bacterial culture broths as an indicator; for e.g.,in Listeria culture. The esculin reaction in demi-Fraser (DF), UVM, andFraser broth base, (which a Listeria-selective enrichment broth bases),is highlighted as a highly likely contributor to assay inhibition. Inthis reaction esculin

is hydrolyzed by specific bacteria to 6,7 dihydroxycoumarin (aesculetin)and glucose. The aesculetin:

then complexes with ferric ions to form a black complex. Aesculetin isin the coumarin family of drugs and coumarins are known to modify theactivity of DNA acting enzymes.

As used herein, the meaning of “surfactant” is the broadest definitionthat is readily recognized by a person of ordinary skill in the art.That is, surfactants are wetting agents that lower the surface tensionof a liquid and/or lower the interfacial tension between two liquids. Asurfactant that does not have a positive or negative charge in water,yet is soluble in water, is a “non-ionic surfactant”.

As used herein, “nonionic surfactant” refers to a surfactant moleculewhose polar group is not electrically charged. Combinations of two ormore non-ionic surfactants are encompassed within the term “non-ionicsurfactant”. In certain embodiments, one or more surfactants may beused.

As used herein, polyvinylpyrrolidone (PVP) is a water-soluble polymermade from the monomer N-vinylpyrrolidone. Polyvinylpolypyrrolidone(PVPP) is a highly cross-linked modification of PVP. As describedherein, polyvinylpyrrolidone, or a modification thereof, can be includedin an amplification reaction mixture so as to reduce or eliminateinhibitory substances. A modified PVP includes, but is not limited topolyvinylpolypyrrolidone (PVPP), which is an insoluble highlycross-linked modification of PVP. It will be understood that disclosureherein related to PVP can be adapted to PVPP.

In an embodiment, the composition may comprises a non-ionic polymericfluorochemical surfactant which belongs to a class of coating additiveswhich provide low surface tensions and exhibits good thermal stabilitywhen used in thermal processing applications. A non-ionic polymericfluorochemical surfactant as per certain embodiments of the presentdisclosure may be FC-4430 which is 3M™ Novec™ fluorosurfactant.

As used herein the terms “ethylene glycol tetraacetic acid” and “EGTA”refer to ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraaceticacid, a chelating agent. EGTA is a colorless solid which has loweraffinity for magnesium, making it more selective for calcium ions. EGTAis useful for making buffer solutions to chelate calcium ions whencalcium ions are less concentrated than magnesium, as found in livingcells. EGTA is also useful in enzyme assays.

As used herein the term “cell lysis” refers to a process of releasingmaterials in a cell by disrupting the cell membrane, and in particular,a process of extracting intracellular materials from a cell to isolateDNA or RNA before amplification, such as PCR, LAMP BART methods andlikewise.

According to an embodiment of the present disclosure, cell lysis may bedone by thermal methods. The thermal method may be properly selected bythose skilled in the art according to the form of cell sample andcharacteristics of reaction vessel.

As used herein, the term “microorganism” or “microbe” refers to anymicroscopic organism, which may be a single cell or multicellularorganism. The term is generally used to refer to any prokaryotic oreukaryotic microscopic organism capable of growing and reproducing in asuitable culture medium, including without limitation, one or more ofbacteria. Microorganisms encompassed by the scope of the presentinvention includes prokaryotes, namely the bacteria and archaea; andvarious forms of eukaryotes, comprising the protozoa, fungi, algae andthe like. As used herein, the term “culture” or “growth” ofmicroorganisms refers to the method of multiplying microbial organismsby letting them reproduce in predetermined culture media underconditions conducive for their growth. More particularly it is themethod of providing a suitable culture medium and conditions tofacilitate at least one cell division of a microorganism. Culture mediamay be solid, semisolid or liquid media containing all of the nutrientsand necessary physical growth parameters necessary for microbialgrowth.ae etc. and also includes viruses. The term “targetmicroorganism” refers any microorganism that is desired to be detected.

As used herein, the term “enrichment” refers to the culture method ofselectively enriching the growth of a specific microorganism byproviding medium and conditions with specific and known attributes thatfavors the growth of that particular microorganism. The enrichmentculture's environment will support the growth of a selectedmicroorganism, while inhibiting the growth of others.

The use of conventional DNA-based methods is to some extent restrictedby the presence of inhibitors. The occurrence of such so calledinhibitors, which comprises all substances that have a negative effecton the nucleic acid proliferation reactions, is one of the drawbacks ofgenetic testing. These inhibitors can originate from the sample itselfor may be introduced during sample processing or nucleic acidextraction. The consequence of a partly or total inhibition of thenucleic acid proliferation reactions is a decreased sensitivity orfalse-negative results, respectively.

Despite the availability of numerous genetic based methods, there is nosingle rapid, sensitive, inexpensive and less laborious method toefficiently and rapidly reduce or eliminate the inhibition of thenucleic acid amplification of the intended target. To quickly determinethe presence of pathogen in targeted sample, there is a need to developa reliable and accurate assay method which can cater to the increasingneed of finding faster, accurate and less time consuming and lesslaborious assay techniques.

In competitive PCR systems a challenge to ease of use is the inclusionof a protease step. This protease step is used for reducing sampleinhibition by digesting food protein (especially red meats) as well aslysing cells. Surprisingly, it has been found that a composition and amethod as per the present disclosure eliminates the need for proteasinga sample by using ferric ammonium citrate to neutralize inhibitoryproteins. Advantageously the present disclosure eliminates the step ofisolation/purification which makes the current assay method faster andsimpler.

The present disclosure describes the composition and method of nucleicacid amplification which eliminates the need to protease or otherwisereduces background protein from the sample. This in turn also leads tothe elimination of one of the assay steps of the conventional nucleicacid amplification methods.

The present disclosure generally relates to novel compositions (e.g.,aqueous compositions) and methods for nucleic acid proliferation of asample which comprises a cell lysis step and nucleic acid amplificationstep without an isolation/purification step such as chromatography,centrifugation and likewise in between.

A composition of the present disclosure is typically used as an aqueoussolution comprising the respective chemical components. Thus, in anyembodiment, a composition of the present disclosure comprises water.Alternatively, a composition of the present disclosure can be mixed withan aqueous liquid (e.g., water or a sample containing water) to form amixture to be used according to the present disclosure. A predeterminedvolume of the aqueous composition can be mixed with a predeterminedamount (e.g., volume) of a sample to form a mixture that is treated(e.g., by heating) to lyse any microorganisms, if present, in thesample. A portion of the resulting lysate can be used in a nucleicamplification process to detect nucleic acid sequences that indicate apresence of one or more target microorganisms in the original sample.

Typically, the sample (e.g., ground beef, a carcass rinse, processwater, residue from an environmental (e.g., food-processing equipment)swab or sponge) is suspended in an aqueous liquid (e.g., water or abuffer). Thus, a first predetermined volume of the aqueous sample ismixed with the composition or with a second predetermined volume of anaqueous solution comprising the composition of the present disclosure toform the mixture that is subjected to a lysis treatment. Accordingly,each component of the composition of the present disclosure is presentin the aqueous composition at a concentration that takes into accountthe dilution that occurs when the sample is mixed with the composition.

According to the present disclosure, the ratio of the firstpredetermined volume to the volume of the mixture formed by mixing thefirst and second predetermined volumes is less than or equal to 1:10. Inany embodiment, the ratio of the first predetermined volume to thevolume of the mixture formed by mixing the first and secondpredetermined volumes is about 1:10 to about 1:300. In any embodiment,the ratio of the first predetermined volume to the volume of the mixtureformed by mixing the first and second predetermined volumes is about1:10, about 1:20, about 1:25, about 1:30, about 1:40, about 1:50, about1:100, about 1:200, about 1:250, or about 1:300.

Accordingly, the present disclosure provides a composition (e.g., anaqueous liquid composition) comprising an organic iron-chelatingreagent, ferric iron, and a non-ionic surfactant at a pH of about8.45-8.85. The composition can be used in nucleic acid amplificationmethods to eliminate sample inhibition of the nucleic acid amplificationreaction. Optionally, the composition can comprise polyvinylpyrrolidone.

In any embodiment, a suitable pH for the composition is at least 8.45.In any embodiment the pH may be within the range of 8.45 to 8.85. Incertain embodiments, a pH of 8.65 to 8.75 may be used. In otherembodiments, a pH of 8.75, to 8.85 may be used.

The organic iron-chelating reagent has a predefined affinity constantfor ferric (Fe⁺³) iron ions. In deionized water at pH 8.45 and 20° C.,the organic iron-chelating reagent has an affinity constant greater thanor equal to 10^(4.2) with respect to ferric iron ions. The organiciron-chelating reagent also has a predefined affinity constant formagnesium (Mg⁺²) ions. In deionized water at pH 8.45 and 20° C., theorganic iron-chelating reagent has an affinity constant less than10^(3.8) with respect to magnesium ions. Thus, in the aqueouscomposition of the present disclosure at pH 8.45, the organiciron-chelating reagent has a higher affinity for ferric iron ions thanfor magnesium ions.

Suitable organic iron-chelating reagents include organic molecules. Inany embodiment, the organic iron-chelating reagent is water-soluble. Inany embodiment, the organic iron-chelating reagent comprises a pluralityof carboxylate groups. Non-limiting examples of suitable organiciron-chelating reagents include ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA);N,N,N′,N′-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN);1,2-bis(o-aminophenoxy)ethane-N,N,N′N′-tetraacetic acid (BAPTA);N-(2-hydroxyethyl) ethylenediamine-N,N′,N′-triacetic acid (HEDTA); andsalts thereof.

In any embodiment, the composition further comprises ferric iron.Accordingly, an aqueous composition of the present disclosure maycomprise ferric iron ions. In any embodiment, the ferric iron can beprovided in the composition by ferric ammonium citrate.

In any embodiment, ferric iron can be present in an aqueous liquidcomposition according to the present disclosure at a concentration offerric ions (i.e., dissolved ferric iron) of about 55 μM-385 μM. Incertain embodiments the concentration of ferric ions may be at least 110μM, in certain other embodiments it may be at least 165 μM. In otherembodiments the concentration of ferric ions may be at least 220 μM andin other embodiments it may be at least 275 μM or at least 330 μM.According to the present disclosure, in an aqueous wherein the organiciron-chelating reagent comprises EGTA, the composition has a molar ratioof dissolved ferric iron/EGTA of about 0.04 to about 0.28. In certainpreferred embodiments, the aqueous composition has a molar ratio ofdissolved ferric iron/EGTA of about 0.14 to about 0.18.

In any embodiment of a liquid (e.g., an aqueous liquid) compositionaccording to the present disclosure, ferric iron (provided as ferricammonium citrate) is present to yield (when the composition is mixedwith a sample) a concentration of ferric ions of about 50 μM-350 μM. Incertain embodiments the concentration of ferric ions may be at least 100μM, in certain other embodiments it may be at least 150 μM. In otherembodiments the concentration of ferric ions may be at least 200 μM, andin other embodiments it may be at least 250 μM or at least 300 μM.According to the present disclosure, in an aqueous wherein the organiciron-chelating reagent comprises EGTA, the composition has a molar ratioof Fe³⁺/EGTA of about 0.04 to about 0.28. In certain preferredembodiments, the composition has a molar ratio of Fe³⁺/EGTA of about0.14 to about 0.18.

In an embodiment, the composition of present disclosure comprises atleast one non-ionic surfactant. Accordingly, the composition maycomprise one or more of any non-ionic surfactant. Preferably, thenon-ionic surfactant has a Hydrophilic-lipophilic balance of about 11 toabout 16. Surfactants with a Hydrophilic-lipophilic balance in thisrange permit sufficient activity of the DNA polymerases in PCR and LAMPnucleic acid amplification reactions as well as permit sufficientluciferase and ATP sulphurlyase activity in the BART reporter technologyExamples of suitable non-ionic surfactants include, but are not limitedto TRITON™ series of detergents, including, but not necessarily limitedto, TRITON X-100 (t-octylphenoxypolyethoxyethanol) and its derivatives,TRITON X-114, TRITON X-405, TRITON X-101, TRITON N-42, TRITON N-57,TRITON N-60, TRITON X-15, TRITON X-35, TRITON X-45, TRITON X-102, TRITONX-155, TRITON X-165, TRITON X-207, TRITON X-305, TRITON X-705-70 andTRITON B-1956; sorbitan fatty acid ester, Polyoxyethylene (POE)sorbitanfatty acid ester (e.g., Tween), POE alkyl ether (e.g., Brij),nonylphenol, lauryl alcohol, polyethylene glycol,polyoxyethylene.polyoxypropylene block polymer, POE alkyl amine, and POEfatty acid bisphenyl ether and fluorosurfactants such as 3M Novec™engineered liquid surfactants FC-4430 and FC4432, and Dow chemical FSseries fluorosurfactants, for example.

In any embodiment of a liquid (e.g., an aqueous liquid) compositionaccording to the present disclosure, the concentration of such asurfactant in the composition is not particularly limited, as long asthe beneficial effects of the present invention (i.e., with respect tofacilitation of nucleic acid amplification) can be achieved. In anyembodiment, a composition of the present disclosure comprises about0.005% (w/v) to about 0.3% (w/v) surfactant. Accordingly, in anyembodiment, a composition of the present disclosure comprises up toabout 0.3% (w/v) surfactant. In certain embodiments the concentration ofsurfactant may be at least 0.01% (w/v) and in certain other embodimentsit may be at least 0.025% (w/v) and in another embodiment it is 0.032%(w/v).

Optionally, in any embodiment of the present disclosure,polyvinylpyrrolidone (PVP) with a nominal molecular weight of 30 KDa to1.3 MDa may be used. In one aspect of the disclosure, PVP has a nominalmolecular weight is 360 KDa.

In an embodiment of the present disclosure, polyvinylpyrrolidone may beincluded in the composition when low amounts of the surfactant is used.In an embodiment of a liquid (e.g., an aqueous liquid) compositionaccording to the present disclosure, the composition (before adding thesample) may comprise 0% w/v up to about 0.0473% w/v PVP. When thenonionic surfactant is present in the composition at a concentration of0.0055% to 0.011% w/v, the composition may comprise about 0.011% w/v toabout 0.0473% w/v PVP. Each of the above concentrations apply also to amodified PVP.

In any embodiment of the present disclosure, polyvinylpyrrolidone may beincluded in the composition when low amounts of the surfactant is used.In an embodiment of a liquid (e.g., an aqueous liquid) compositionaccording to the present disclosure, the composition (after adding thesample) may comprise 0% w/v up to about 0.043% w/v PVP. When thenonionic surfactant is present in the composition at a concentration of0.005% to 0.01% w/v, the composition may comprise about 0.01% w/v toabout 0.043% w/v PVP. Each of the above concentrations apply also to amodified PVP.

In certain embodiments of the disclosure (e.g., when the nonionicsurfactant concentration is >0.01% w/v), polyvinylpyrrolidone may not beincluded in the composition.

In any embodiment of the present disclosure, the organic iron-chelatingreagent comprises EGTA. In any embodiment, the organic iron-chelatingreagent can be provided to the composition in the form of a salt. Incertain embodiments, the composition may include, for example, a sodiumsalt of EGTA. In another embodiment of the present disclosure, thecomposition may include, for example, a potassium salt of EGTA.

A composition according to the present disclosure may comprise Fe³⁺(e.g., provided via ferric ammonium citrate) and ethylene glycoltetraacetic acid (e.g., provided via a salt (e.g., a monovalent cationsalt) of ethylene glycol tetraacetic acid). Thus, in the composition,there can exist a molar ratio of ethylene glycol tetraacetic acid andFe³⁺. In any embodiment, the molar ratio of ethylene glycol tetraaceticacid to F³⁺ is about 0.04 to about 0.28.

In any embodiment of a liquid (e.g., an aqueous liquid) compositionaccording to the present disclosure, the EGTA may be present at aconcentration of 0.5 mM to 5 mM. In any embodiment, the molar ratio ofmetal ion to EGTA is at least 0.6.

According to one embodiment of the present disclosure, a sample may betested directly, or may be prepared or processed in some manner prior totesting. For example, a sample may be processed to enrich anycontaminating microbe and may be further processed to separate and/orlyse microbial cells/viral cells/fungal cells contained therein. Lysedmicrobial cells from a sample may be additionally processed or preparesto separate and/or extract genetic material from the microbe foranalysis to detect and/or identify the contaminating microbe. Someembodiments refer to “a nucleic acid in a sample” or a “sample derivednucleic acid” and refer to a nucleic acid comprised in a sample orobtained from a sample. Such nucleic acids can be tested by methods andusing compositions described herein.

In certain embodiments of the present disclosure, a sample may besubjected to separation to initially separate microbes of interest fromother microbes and other sample components. For example, for complexfood samples with complex components separation methods can be used toseparate microorganisms from food. Separated microbes from samples mayalso be enriched prior to analysis. Analysis of a sample may include oneor more molecular methods. For example, according to some exemplaryembodiments of the present disclosure, a sample may be subject tonucleic acid amplification (for example by LAMP-BART) using appropriateoligonucleotide primers that are specific to one or more microbe nucleicacid sequences that the sample is suspected of being contaminated with.Amplification products may then be further subject to testing withspecific probes (or reporter probes) to allow detection of microbialnucleic acid sequences that have been amplified from the sample. In someembodiments, if a microbial nucleic acid sequence is amplified from asample, further analysis may be performed on the amplification productto further identify, quantify and analyze the detected microbe(determine parameters such as but not limited to the microbial strain,pathogenicity, quantity etc.).

The present disclosure is generally provides a nucleic acidamplification method, said method comprising a) contacting anycomposition (e.g., aqueous composition) according to the presentdisclosure with a target sample to form an aqueous mixture wherein themixture has a pH of about 8.45 to 8.85; b) subjecting the aqueousmixture of step a) to thermal lysis; and c) subsequent to step b),subjecting a portion of the aqueous mixture to isothermal nucleic acidamplification.

In any embodiment of the present disclosure, a nucleic acidamplification method, comprises a) contacting any composition (e.g.,aqueous composition) according to the present disclosure with a targetsample to form an aqueous mixture wherein the aqueous mixture has a pHof about 8.45 to 8.85; b) subjecting the aqueous mixture of step a) tothermal lysis; and c) subsequent to step b), subjecting the aqueousmixture to isothermal nucleic acid amplification, wherein said methodeliminates the inhibition of nucleic acid amplification caused byinterfering components present in the sample.

The present disclosure is generally provides a nucleic acidamplification method, said method comprising a) contacting anycomposition (e.g., aqueous composition) according to the presentdisclosure with a sample to form an aqueous mixture; wherein the aqueousmixture has a pH of about 8.45 to 8.85; b) subjecting the aqueousmixture of step a) to thermal lysis; and c) subsequent to step b),subjecting a portion of the aqueous mixture to isothermal nucleic acidamplification.

The amplification methods used in a method according to the presentdisclosure may be performed isothermally. Isothermal techniques includebut not limited loop-mediated isothermal amplification (LAMP), stranddisplacement amplification (SDA), nucleic acid sequence-basedamplification (NASBA). The reaction proceeds at a constant temperatureusing strand displacement reactions. Amplification can be completed in asingle step, by incubating the mixture of samples, primers, DNApolymerase with strand displacement activity, and substrates at aconstant temperature. In addition to steps or reactions that increasethe number of copies of a target nucleic acid sequence, theamplification methods further may include steps or reactions to detectthe amplified target nucleic acid sequence. Such detection steps orreactions are well known to a person having ordinary skill in the artand include, for example, bioluminescent real-time reporter (BART) stepsor reactions.

In an embodiment of the present disclosure, the isothermal amplificationreaction is a Loop-mediated isothermal amplification (LAMP-BART) method.LAMP can amplify DNA with high specificity, efficiency and rapidityunder isothermal condition. The LAMP method requires a Bst DNApolymerase and set of four to six specific designed primers thatrecognize a total of six distinct sequences of the target DNA and withstrand displacement activity. In Loop-mediated isothermal amplification(LAMP), target-specific amplification is achieved by the use of 4 to 6different primers specifically designed to recognize 6 to 8 distinctregions on the target gene, respectively. Such methods typically amplifynucleic acid copies 10⁹-10¹⁰ times in 15-60 minutes. In addition, thepresence of, for example, ATP-sulfurylase, adenosine-5′-O-persulfate,luciferin, and luciferase in the amplification reaction permitsdetection of a LAMP-mediated amplification reaction via bioluminescence(i.e., the BART reaction).

In addition to the primers, LAMP-BART techniques use Tris, sulfatecompounds (such as MgSO₄, NH₄SO₄) and potassium chloride to maintainenzyme functionality. Thus, such compounds are act as enhancers tofacilitate the LAMP-BART coupled reaction. Tris is an organic compound(more formally known as tris (hydroxymethyl) aminomethane, with theformula (HOCH₂)₃CNH₂). Strand displacement techniques, such as LAMP, useTris as a buffer, which maintain the reaction at the optimal pH.

Compositions (e.g., aqueous compositions) of the present disclosureoptionally can comprise an indicator dye to monitor the approximatetemperature of an aqueous solution comprising the composition.Advantageously, the indicator dye can provide a first visual indication(e.g., a first observable color) to indicate that an aqueous mixturecomprising the composition has reached a temperature (e.g., about 100°C.) approximately in a range that is suitable for thermal lysis ofmicrobial cells in contact with the composition In addition, theindicator dye can provide a second visual indication (e.g., a secondcolor) to indicate that the aqueous mixture comprising the compositionhas cooled to a temperature (e.g., ≤° C.) that is suitable to remove aportion of the mixture and place it into a nucleic acid amplificationreaction. Certain pH indicators (e.g., those having a transition rangethat at least partially extends between a pH of about 8.8 and about 7.2)can be readily monitored as the pH of the aqueous mixture changes duringheating and cooling steps.

Suitable visible dyes include, for example, Cresol Red, which has areddish-purple color when pH is higher than 8.8 and a yellow color whenpH is less than 7.2

In any of the embodiments of the present disclosure, the indicator dyemay be cresol red.

Using LAMP, the target nucleic acid sequence is amplified at a constanttemperature of 60° C. to 65° C. using either two or three pairs ofprimers and a polymerase with high strand displacement activity inaddition to a replication activity. The loop-mediated isothermalamplification (LAMP) reaction is a highly specific, sensitive,isothermal nucleic acid amplification reaction. LAMP employs a primerset of four essential primers, termed forward inner primer (FIP),backward inner primer (BIP), forward displacement primer (F3) andbackward displacement primer (B3). These four different primers are usedto identify 6 distinct regions on the target gene, which adds highly tothe specificity. Due to the specific nature of the action of theseprimers, the amount of DNA produced in LAMP is considerably higher thanPCR-based amplification. Furthermore, two optional primers can beincluded which effectively accelerate the reaction; these are termedforward loop primer (LF) and backward loop primer (LB). The innerprimers (FIP and BIP) contain sequences of the sense and antisensestrands of the target DNA, while the displacement primers (F3 and B3)and the loop primers (LF and LB) each contain a single target sequence.In total, eight target sequences are recognized when including loopprimers (LF and LB) in the reaction. A DNA polymerase is used to amplifythe target sequence of interest. Many different DNA polymerases may beused including engineered DNA polymerases not found in nature, the mostcommon being the Bst DNA polymerase while the Geobacillus sp. largefragment (GspSSD) DNA polymerase is used less often.

The LAMP reaction is initiated by DNA synthesis primed by the innerprimers (FIP and BIP). This is followed by DNA synthesis primed by adisplacement primer (F3 or B3) which releases a single-stranded DNA.This single-stranded DNA serves as template for DNA synthesis primed bythe second inner and displacement primers that hybridize to the otherend of the target. This produces a stem-loop DNA structure. Insubsequent LAMP cycling, one inner primer hybridizes to the loop on theproduct and initiates displacement DNA synthesis. This yields theoriginal stem-loop DNA and a new stem-loop DNA with a stem twice aslong. The cycling reaction continues with accumulation of around 10⁹copies of target in less than an hour. The inclusion of one or two loopprimers (LF and/or LB) accelerates the LAMP reaction by hybridizing tothe stem-loops, except for the loops that are hybridized by the innerprimers, and prime strand displacement DNA synthesis. A variety of LAMPamplification detection methods exist. Non-specific target detection maybe obtained through visual identification of a turbid sample asmagnesium pyrophosphate precipitates in a positive LAMP reaction. Forbetter visibility of a positive reaction, various agents, such ashydroxy naphthol blue or calcein, may be added to the reaction.Alternatively, fluorescent detection may be achieved using a DNAintercalating dye, such as cresol red, SYBR green, Picogreen orpropedium iodide, which is added to the reaction reagent or added afterthe completion of the reaction for end point analysis.

In an embodiment, the present disclosure provides a method that includescontacting a sample suspended in the composition as per the presentdisclosure with components of an isothermal nucleic acid amplificationreaction for a target nucleic acid species, thereby providing anamplification reaction mixture; incubating the amplification reactionmixture under conditions sufficient for the isothermal nucleic acidamplification reaction to proceed, thereby providing a product; anddetermining whether an indicator of the target nucleic acid species ispresent in the product.

In another embodiment, the disclosure features a method that includesperforming an isothermal reaction of an amplification reaction mixtureto provide a product, the mixture comprising a lysate which includes thesample mixed with the composition as per the present disclosure andcomponents of a nucleic acid amplification reaction for a target nucleicacid species; and determining whether an indicator of the target nucleicacid species is present in the product.

The components of an isothermal amplification reaction may be providedin a solution and/or in dried (e.g., lyophilized) form. When one or moreof the components are provided in dried form, a resuspension orreconstitution buffer may be also be used. Alternatively, after formingan aqueous mixture comprising the sample and the composition of thepresent disclosure and, after subjecting the aqueous mixture to athermal lysis procedure, the aqueous mixture can be used to reconstitutethe components of the isothermal reaction.

Based on the particular type of amplification reaction, the reactionmixture can contain buffers, salts, nucleotides, and other components asnecessary for the reaction to proceed. The reaction mixture may beincubated at a specific temperature appropriate to the reaction.

The target nucleic acid maybe a nucleic acid present in an animal (e.g.,human), plant, fungal (e.g., yeast), protozoan, bacterial, or viralspecies. For example, the target nucleic acid may be present in thegenome of an organism of interest (e.g., on a chromosome) or on anextra-chromosomal nucleic acid. In some embodiments, the target nucleicacid is an RNA, e.g., an mRNA. In particular embodiments, the targetnucleic acid is specific for the organism of interest, i.e., the targetnucleic acid is not found in other organisms or not found in organismssimilar to the organism of interest.

The present disclosure has manifold applications in various fields thatrequire method of detecting a microorganism in a sample wherein thecomposition may be used as a suspending medium into which sample is heldduring a thermal lysis step and composition may be used as the aqueousmedium in which a lyophilized pellet of LAMP-BART nucleic acidamplification reagents are dissolved and allowed to react.

The present disclosure effortlessly allows the user to merely contactthe sample with the composition comprising an organic iron-chelatingreagent, ferric iron, and a non-ionic surfactant, EGTA (or a monovalentsalt thereof), and optionally polyvinylpyrrolidone at a pH of about 8.4to 8.85 to form an aqueous mixture; subsequently to subject the aqueousmixture to a lysis procedure (e.g., a thermal lysis procedure); and,after cooling the aqueous mixture, to subject the aqueous mixture toisothermal nucleic acid amplification reaction for detection ofmicroorganisms.

In another embodiment, the present disclosure provides kits. In general,the kits comprises an organic iron-chelating reagent according to thepresent disclosure, ferric iron, a non-ionic surfactant, and optionallypolyvinylpyrrolidone.

In an embodiment, the kit comprises an organic iron-chelating reagent,ferric iron, and a non-ionic surfactant. The organic iron-chelatingreagent has a first affinity constant greater than or equal to 10^(4.2)with respect to ferric iron and a second affinity constant less than10^(3.8) with respect to magnesium, wherein the first affinity constantand the second affinity constant are determined in deionized water at pH8.45 and 20°.

In any embodiment of the kit, the organic iron-chelating reagent cancomprise a plurality of carboxylate groups. In any of the aboveembodiments, the kit further can comprise2-hydroxypropane-1,2,3-tricarboxylate (citric acid). In any of the aboveembodiments of the kit, the organic iron-chelating reagent can compriseEGTA, wherein a molar ratio of the ferric iron to the EGTA is about 0.04to about 0.28.

In another embodiment, a kit may further comprise a component selectedfrom the group consisting of fluorosurfactant, indicator dye,preservative, buffering agents and enhancers of LAMP-BART reaction andcombinations thereof. In any embodiment of the kit, any one or more ofthe foregoing components may be present in the kit in the composition.The kit may comprise at least one primer, such as two primers, foramplification of a target nucleic acid. It also may include at least oneother primer for amplification of a target nucleic acid, which can be,but is not necessarily, the same nucleic acid (and even the samesequence within the same nucleic acid) that is the target for one ormore other primer(s) in the kit. In some embodiments, the kits comprisetwo or more primers for amplifying one or more unique genomic sequences.

In another embodiment, the kits comprise the components (i.e., thecomposition, the components thereof, the fluorosurfactant, the indicatordye, the preservative, the buffering agent and/or the enhancer of theLAMP-BART reaction) in a single package or in more than one packagewithin the same kit. Where more than one package is included within akit, each package can independently contain a single component ormultiple components, in any suitable combination. As used herein, acombination of two or more packages or containers in a single kit isreferred to as “in packaged combination”.

In any embodiment of the kit, any one or more of the organiciron-chelating reagent, the ferric iron, the polyvinylpyrrolidone, thenon-ionic surfactant, the fluorosurfactant, the indicator dye, thepreservative, the buffering agent, or enhancer is disposed in an aqueoussolution. In any embodiment, the aqueous solution can have a pH of about8.45 to 8.85.

The kits and containers within the kits may be fabricated with any knownmaterial. For example, the kits themselves may be made of a plasticmaterial or cardboard. The containers that hold the components may be,for example, a plastic material or glass. Different containers withinone kit may be made of different materials. In embodiments, the kit cancontain another kit within it.

The kit of the present disclosure may comprise one or more componentsuseful for amplifying target sequences. In embodiments, some or all ofthe reagents and supplies necessary for performing LAMP-BART method areincluded in the kit. Non-limiting examples of reagents are buffers(e.g., a buffer containing Tris, HEPES, and the like), salts, and atemplate-dependent nucleic acid extending enzyme (such as a thermostableenzyme, such as Taq polymerase), a buffer suitable for activity of theenzyme, and additional reagents needed by the enzyme, such as dNTPs,dUTP, and/or a UDG enzyme. In embodiments, the kit comprises enhancerssuch as potassium chloride and ammonium sulfate to facilitate theenzymatic reactions. A non-limiting example of supplies is reactionvessels (e.g., microfuge tubes).

The kit has the advantages of high sensitivity, high specificity, easeof operation, capability of judging a result through naked eyes and thelike.

Exemplary Embodiments

Embodiment A is a composition, said composition comprising:

an organic iron-chelating reagent;

ferric iron;

a non-ionic surfactant at a concentration greater than or equal to0.005% (mass/volume); and

wherein the composition has a pH of about 8.45 to 8.85;

wherein the organic iron-chelating reagent has a first affinity constantgreater than 10^(4.2) with respect to ferric iron and a second affinityconstant less than 10^(3.8) with respect to magnesium, wherein the firstaffinity constant and the second affinity constant are determined indeionized water at pH 8.45 and 20° C.

Embodiment B is the composition of Embodiment A, said compositionfurther comprising water, wherein the non-ionic surfactant is present inthe composition at a concentration greater than or equal to 0.005%(mass/volume).

Embodiment C is the composition of Embodiment A or Embodiment B, whereinthe organic iron-chelating reagent comprises a plurality of carboxylategroups.

Embodiment D is the composition of any one of the preceding Embodiments,wherein the organic iron-chelating reagent is water-soluble.

Embodiment E is the composition of Embodiment D, wherein the organiciron-chelating reagent is selected from the group consisting of EGTA;N,N,N′,N′-tetrakis(2-pyridylmethyl)ethane-1,2-diamine;1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid;N-(2-Hydroxyethyl)-ethylenediamine-N,N′,N′-triacetic acid; and saltsthereof.

Embodiment F is the composition of Embodiment E, wherein the organiciron-chelating reagent comprises EGTA, wherein a molar ratio of ferriciron to the EGTA is about 0.04 to about 0.28.

Embodiment G is the composition of Embodiment F, wherein the EGTA ispresent at a concentration of 0.5 mM to 5 mM.

Embodiment H is the composition of Embodiment F or Embodiment G, whereinthe EGTA is present in the composition as a sodium or potassium salt.

Embodiment I is the composition of any one of the preceding Embodiments,further comprising 2-hydroxypropane-1,2,3-tricarboxylate; wherein theorganic iron-chelating reagent and the2-hydroxypropane-1,2,3-tricarboxylate are distinct molecules.

Embodiment J is the composition of any one of the preceding Embodiments,wherein the non-ionic surfactant has a Hydrophilic-lipophilic balance ofabout 11 to about 16.

Embodiment K is the composition of any one of the preceding Embodiments,wherein the nonionic surfactant is selected from the group consisting oft-octylphenoxypolyethoxyethanol, sorbitan fatty acid ester,polyoxyethylene sorbitan fatty acid ester, polyoxyethylene alkyl ether,nonylphenol, lauryl alcohol, polyethylene glycol,polyoxyethylene.polyoxypropylene block polymer, polyoxyethylene alkylamine, and polyoxyethylene fatty acid bisphenyl ether, and a combinationof any two or more of the foregoing nonionic surfactants.

Embodiment L is the composition of any one of the preceding Embodiments,wherein the non-ionic surfactant is present at a concentration up toabout 0.3% mass/volume.

Embodiment M is the composition of any one of Embodiments B through L,wherein the ferric iron is dissolved in the aqueous composition at aconcentration of 50 μM to 300 μM.

Embodiment N is the composition of any one of the preceding Embodiments,further comprising polyvinylpyrrolidone.

Embodiment O is the composition of Embodiment N, polyvinylpyrrolidone ispresent at a concentration of up to 0.043% w/v.

Embodiment P is the composition of any one of the preceding Embodiments,wherein the composition further comprises fluorosurfactant.

Embodiment Q the composition of Embodiment of P, whereinfluorosurfactant is FC-4430™.

Embodiment R is the composition of any one of the preceding Embodiments,wherein the composition further comprises an indicator dye.

Embodiment S is the composition of Embodiment of R, wherein indicatordye is cresol red.

Embodiment T is the composition of any one of the preceding Embodiments,wherein the composition further comprises a preservative.

Embodiment U is the composition of Embodiment of T wherein preservativeis methylisothiazolinone.

Embodiment V is the composition of any one of the preceding Embodiments,for use in detecting microorganism in a sample.

Embodiment W is the composition of any one of the preceding Embodiments,wherein the sample is a food sample, clinical sample or a culture broth.

Embodiment X is the composition of Embodiment of W, wherein food samplecomprises protein.

Embodiment Y is the composition of Embodiment X, wherein protein isferritin.

Embodiment Z is the composition of any one of the preceding Embodiments,wherein the sample comprises a culture broth.

Embodiment AA is the composition of Embodiment Z, wherein the samplecomprises esculin.

Embodiment AB is an isothermal nucleic acid amplification method, saidmethod comprising

-   a) contacting the composition of any one of Embodiments B through AA    with a target sample to form an aqueous mixture;-   b) subjecting the aqueous mixture of step a) to a thermal lysis    process; and-   c) after step b), subjecting the aqueous mixture to isothermal    nucleic acid amplification process.

Embodiment AC is the method of Embodiment AB, for eliminating inhibitionof nucleic acid amplification caused by interfering components presentin the sample.

Embodiment AD is the method of Embodiment AB or Embodiment AC, whereinthe isothermal nucleic acid amplification method is a loop-mediatedisothermal amplification (LAMP) method.

Embodiment AE is the method of any one of the Embodiments AB through AD,wherein the sample is a food sample, clinical sample or a culture broth.

Embodiment AF is the method of Embodiment AE, wherein the food samplecomprises protein.

Embodiment AG is the method of Embodiment AF, wherein protein isferritin.

Embodiment AH is the method of any one of the Embodiments AB through AG,wherein the sample is culture broth.

Embodiment AI is the method of Embodiment AH, wherein the samplecomprises esculin.

Embodiment AJ is the method of any one of the Embodiments AB through AI,wherein the sample is incubated in a culture broth at about 41.5° C.prior to step a).

Embodiment AK is the method of any one of the Embodiments AB through AJ,wherein composition further comprises a buffering agent, and an enhancerfor facilitating LAMP-BART reaction.

Embodiment AL is the method of Embodiment AK, wherein the enhancer isselected from the group consisting of potassium chloride, ammoniumsulfate, magnesium sulfate heptahydrate and combinations thereof.

Embodiment AM is the method of Embodiment AK or Embodiment AL, whereinthe buffering agent comprises Tris base.

Embodiment AN is the method of any one of the Embodiments AB through AM,wherein the subjecting the aqueous mixture to a thermal lysis processcomprises heating the aqueous mixture to about 100° C. for about 15minutes and subsequently cooling the mixture to about 40° C. for about 5minutes.

Embodiment AO is the method of Embodiment AN, wherein, after cooling themixture to about 40° C., the method further comprises contacting themixture with a reagents for LAMP-BART isothermal amplification.

Embodiment AP is a kit, comprising:

ferric iron;

an organic iron-chelating reagent; and

a non-ionic surfactant;

wherein the organic iron-chelating reagent has a first affinity constantgreater than or equal to 10^(4.2) with respect to ferric iron and asecond affinity constant less than 10^(3.8) with respect to magnesium,wherein the first affinity constant and the second affinity constant aredetermined in deionized water at pH 8.45 and 20° C.

Embodiment AQ is the kit of Embodiment AP, wherein the organiciron-chelating reagent comprises a plurality of carboxylate groups.

Embodiment AR is the kit of Embodiment AP, wherein the organiciron-chelating reagent is selected from the group consisting of ethyleneglycol tetraacetic acid;N,N,N′,N′-tetrakis(2-pyridylmethyl)ethane-1,2-diamine;1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid;N-(2-Hydroxyethypethylenediamine-N,N′,N′-triacetic acid; and saltsthereof.

Embodiment AS is the kit of any one of Embodiments AP through AR,further comprising polyvinylpyrrolidone.

Embodiment AT is the kit of any one of Embodiments AP through AS,wherein non-ionic surfactant is selected from the group consisting oft-octylphenoxypolyethoxyethanol, sorbitan fatty acid ester,polyoxyethylene sorbitan fatty acid ester, polyoxyethylene alkyl ether,nonylphenol, lauryl alcohol, polyethylene glycol,polyoxyethylene.polyoxypropylene block polymer, polyoxyethylene alkylamine, polyoxyethylene fatty acid bisphenyl ether, and a combination ofany two or more of the foregoing nonionic surfactants.

Embodiment AU is the kit of any one of any one of Embodiments AP throughAT, wherein organic iron-chelating reagent comprises EGTA, BAPTA, HEDTA,TPEN, or a monovalent cationic salt of any of the foregoing organiciron-chelating reagents.

Embodiment AV is the kit of Embodiment AU, wherein the monovalentcationic salt is selected is selected from a sodium salt and a potassiumsalt.

Embodiment AW is the kit of any one of the Embodiments AP through AV,wherein the kit further comprises a fluorosurfactant.

Embodiment AX is the kit of Embodiment AW, wherein fluorosurfactant isFC-4430™.

Embodiment AY is the kit of any one of Embodiments AP through AX,wherein the kit further comprises an indicator dye.

Embodiment AZ is the kit of Embodiment AY, wherein the indicator dye iscresol red.

Embodiment BA is the kit of any one of the Embodiments AP through AZ,wherein the kit further comprises a preservative.

Embodiment BB is the kit of Embodiment BA, wherein the preservative ismethylisothiazolinone.

Embodiment BC is the kit of any one of the Embodiments AP through BB,wherein the kit further comprises a buffering agent.

Embodiment BD is the kit of any one of the Embodiments AP through BC,wherein the kit further comprises a buffering agent, and an enhancer forfacilitating LAMP-BART reaction.

Embodiment BE is the kit of Embodiment BD, wherein the enhancer isselected from the group consisting of potassium chloride, ammoniumsulfate, magnesium sulfate heptahydrate and combinations thereof.

Embodiment BF is the kit of Embodiment BE, wherein the buffering agentcomprises Tris base.

Embodiment BG is the kit of any one of the Embodiments AP through BF,wherein at least one of the organic iron-chelating reagent, the ferriciron, the polyvinylpyrrolidone, and the non-ionic surfactant is disposedin an aqueous medium.

Embodiment BH is the kit of Embodiment BG, wherein the aqueous mediumhas a pH of about 8.45 to 8.85.

Embodiment BI is the kit of any one of the Embodiments AP through BH,wherein the kit comprises instructions for use.

EXAMPLES

Objects and advantages of this invention are further illustrated by thefollowing examples, but the particular materials and amounts thereofrecited in these examples, as well as other conditions and details,should not be construed to unduly limit this invention. Unless otherwiseindicated, all parts and percentages are on a weight basis, all water isdistilled water, and all molecular weights are weight average molecularweight.

Example 1—the Effect of a Composition Comprising Ferric Iron and anOrganic Iron-Chelating Reagent in the LAMP-BART Reaction with a SampleContaining Protein

Sample: Raw Meat

25 g of an 80% lean ground beef sample was added to 225 mL ofroom-temperature demi-Fraser (3M) in a Nasco Whirl-Pak® filter bag andthe mixture was stomached for 2 minutes. The bag holding the stomachedmixture was placed in a 37° C. incubator and incubated for 24 hours forenrichment. A 20 μL aliquot of the enriched sample was added intriplicate to 580 μL of four different formulations of lysis solutionscontaining 150, 100, 50, or 0 μM ferric ammonium citrate, respectively.In addition to ferric ammonium citrate, each of the four lysis solutionscontained the reagents as given below in Table 1.

TABLE 1 Base formulation for lysis solutions. Various concentrations offerric ammonium citrate was added to this base formulation as describedin Example 1. Reagents Concentration Potassium chloride 3.190 g/LAmmonium sulphate 1.410 g/L Proclin ® 950(9.5%) 0.526 mL/L PVP 0.430 g/LTRITON X-100 1.000 g/L FC 4430 0.200 g/L Tris base 2.720 g/L EGTA 0.4754g/L Cresol Red 10 mg/L

The tubes of lysis solution were heated on a 100° C. 3M heat block for15 minutes, cooled to ˜40° C. and a 20 μL aliquot was added to a genericmatrix control LMAP-BART pellet (Part No. MDMC96NA, available from 3MCompany; St. Paul, Minn.). The pellet was placed in a MDS instrument(Part No. MDS 100; available from 3M Company; St. Paul, Minn.) andbioluminescence (i.e., the BART reaction) was recorded for 75 minutes.

FIG. 1 demonstrates the inhibition relief in a dose dependent responsewhere increasing Fe⁺³ concentrations leads to faster reaction times. Itdepicts the effect of different concentration of Fe⁺³ on BART lightemission. Light intensity is measured in terms of the Relative LightUnits (RLU) which is directly proportional to the nucleic acidamplification. FIG. 1 shows that Fe⁺³ at 150 μM concentration resultedin the highest RLU peak; Fe⁺³ at 100 μM and 50 μM concentration resultedin smaller peaks; and RLU was minimum in the absence of Fe⁺³ (0 μM). Itwas also noted that the reaction time was much faster with higher Fe⁺³concentration and was slowest in the absence of Fe⁺³ (0 μM).

This is indicative of the fact that the composition of the presentdisclosure causes reduction/elimination in the nucleic acidamplification inhibition caused by the protein present in the raw meatsample.

Example 2—the Effect of a Composition Comprising Ferric Iron and anOrganic Iron-Chelating Reagent in the LAMP-BART Reaction of a SampleContaining Esculin or Esculetin

An overnight culture of Listeria species (LM 7244), in demi-Fraser (3M)was serially diluted (to 10⁻⁶) in four different media as below:

-   -   1. Demi-Fraser (DF)—without FAC    -   2. Demi-Fraser+FAC (100 μL/10 mL)    -   3. Demi-Fraser+6,7 dihydroxycoumarin [aesculetin] (0.52 g/L)    -   4. Demi-Fraser+6,7 dihydroxycoumarin [aesculetin] (0.52 g/L)+FAC        (100 μL/10 mL)

Each of the above media was then incubated at 37° C. for 6 hours. 20 μLof each sample was added to 5804 of the lysis formulation as describedin table −1 above, boiled on the heat block for 15 minutes, and a 204aliquot was added to a 3M™ Molecular Detection System (MDS) 1.0 L.monocytogenes pellet (part no. MDALM96NA; available from 3M Company; St.Paul, Minn.).

FIG. 2 is a plot of “time to peak” (TTP) in the LAMP-BART reaction inrelation to the 4 media tested as above. “TTP” refers to the amount oftime (minutes) to a positive result using the 3M Molecular DetectionSystem. The word “Coumarin”, as used in FIG. 2, refers to6,7,-dihydroxycoumarin, a product of enzymatic hydrolysis of esculin.

This is indicative of the fact that the composition of the presentdisclosure causes reduction/elimination in the nucleic acidamplification inhibition caused by esculin or degradation productsthereof.

Example 3—the Effect of Surfactant Vis a Vis a Composition ComprisingFerric Iron and an Organic Iron-Chelating Reagent in the LAMP-BARTReaction

A sample of 25 g chicken parts and pieces, in Bolton Broth along with 5%laked horse blood was incubated at 41.5° C. for 24 hours:

-   -   1) Triplicate 204 aliquots of the broth culture were added to        separate tubes containing 580 μl of lysis solution    -   2) The tubes were placed in a heat block for 15 minutes    -   3) Afterward, the tube racks were placed on a room temperature        chill block for 5 minutes    -   4) Finally, 20 μL of the lysates were used to reconstitute a MDS        matrix control pellet and the isothermal amplification reaction        was run.

Lysis solution was same as described in table 1, except that the amountof TRITON-X-100 was varied and magnesium sulphate heptahydrate (73.8mg/L) was additionally present. Surfactant and FAC were varied accordingto the concentrations listed in Table 2.

TABLE 2 Experimental Surfactant FAC Condition (ppm) (nM) TTP (min) A 1000 59.5 100 0 75 100 0 75 B 250 0 75 250 0 75 250 0 75 C 320 0 75 320 052.5 320 0 75 D 100 200 20.75 100 200 24.5 100 200 24.5 E 250 200 36.5250 200 43.75 250 200 48.25 F 320 200 38.25 320 200 49.25 320 200 44.75

FIG. 3 is a boxplot of the data described in table 2, which show thatcontrolling the surfactant level below the Critical MicelleConcentration (CMC) of a given surfactant (TRITON X-100 CMC=190 ppm)adds to the anti-inhibitory effect of Ferric ions and leads to enhancedperformance (i.e., faster time to positive result). Without being boundby theory, it was investigated that effect may be due to decreasedprotein solubilization from the raw meat sample. The lower boundary ofeffective surfactant concentration is about 20 ppm. At surfactantconcentrations <20 ppm, the isothermal amplification reaction wasinhibited, possibly due to the amplification/detection assay enzymessticking to tube walls. In addition to ferric ammonium citrate in thelysis buffer, there is a beneficial effect when surfactant levels arekept below the CMC of the surfactant.

The present disclosure, in general, is suitable for use in both researchand diagnostics. That is, the compositions, methods and kits of thepresent disclosure may be used for the purpose of identifying variousnucleic acids or expressed genes, or for other research purposes.Likewise, the compositions, methods and kits can be used to diagnosenumerous diseases or disorders of humans and animals. In addition, theycan be used to identify diseased or otherwise tainted food products(e.g., foods that are infected with one or more pathogenicorganisms/micro-organisms), or the presence of toxic substances ortoxin-producing organisms in a sample. Thus, the compositions andmethods have human health and veterinary applications, as well as foodtesting and homeland security applications.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material cited herein areincorporated by reference. In the event that any inconsistency existsbetween the disclosure of the present application and the disclosure(s)of any document incorporated herein by reference, the disclosure of thepresent application shall govern. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

The invention illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising”, “consisting essentially of”, and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention claimed is:
 1. An aqueous composition for eliminatingsample inhibition in an isothermal nucleic acid amplification reaction,said aqueous composition comprising: an organic iron-chelating reagent;ferric iron; and a non-ionic surfactant at a concentration greater thanor equal to 0.005% (mass/volume); wherein the aqueous composition has apH of about 8.45-8.85; wherein the organic iron-chelating reagent has afirst affinity constant greater than or equal to 10^(4.2) with respectto ferric iron and a second affinity constant less than 10^(3.8) withrespect to magnesium, wherein the first affinity constant and the secondaffinity constant are determined in deionized water at pH 8.45 and 20°C.; and wherein the composition further comprises2-hydroxypropane-1,2,3-tricarboxylate, wherein the organiciron-chelating reagent and the 2-hydroxypropane-1,2,3-tricarboxylate aredistinct molecules.
 2. The aqueous composition of claim 1, wherein theorganic iron-chelating reagent is selected from the group consisting ofethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid;N,N,N′,N′-tetrakis(2-pyridylmethyl)ethane-1,2-diamine;1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid;N-(2-Hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid; and saltsthereof.
 3. The aqueous composition of claim 1, wherein the organiciron-chelating reagent comprises EGTA, wherein a molar ratio of ferriciron to the EGTA is about 0.04 to about 0.28.
 4. The aqueous compositionof claim 1, wherein the non-ionic surfactant has aHydrophilic-lipophilic balance of about 11 to about
 16. 5. The aqueouscomposition of claim 1, wherein the non-ionic surfactant is present at aconcentration up to about 0.3% (mass/volume).
 6. The aqueous compositionof claim 1, wherein the ferric iron is dissolved in the aqueouscomposition at a concentration of about 50 μM to about 350 μM.
 7. Theaqueous composition of claim 1, further comprising polyvinylpyrrolidone.8. The composition of claim 1, further comprising a fluorosurfactant. 9.The composition of claim 1, further comprising an indicator dye.
 10. Anisothermal nucleic acid amplification method, said method comprising: a)contacting the composition of claim 1 with a target sample to form anaqueous mixture; b) subjecting the aqueous mixture of step a) to athermal lysis process; and c) subsequent to step b), subjecting aportion of the aqueous mixture to an isothermal nucleic acidamplification process.
 11. The method of claim 10, wherein isothermalnucleic acid amplification process comprises loop-mediated isothermalamplification.
 12. The method of claim 10, wherein the compositionfurther comprises a buffering agent, and an enhancer for facilitating aLAMP-BART reaction.
 13. The method of claim 10, wherein subjecting themixture to thermal lysis comprises heating the mixture to about 100° C.for about 15 minutes, wherein the cooled lysate is added to a controlLAMP-BART pellet for isothermal amplification.
 14. An aqueouscomposition for eliminating sample inhibition in an isothermal nucleicacid amplification reaction, said aqueous composition comprising: anorganic iron-chelating reagent; ferric iron; and a non-ionic surfactantat a concentration greater than or equal to 0.005% (mass/volume);wherein the aqueous composition has a pH of about 8.45-8.85; wherein theorganic iron-chelating reagent has a first affinity constant greaterthan or equal to 10^(4.2) with respect to ferric iron and a secondaffinity constant less than 10^(3.8) with respect to magnesium, whereinthe first affinity constant and the second affinity constant aredetermined in deionized water at pH 8.45 and 20° C.; and wherein thecomposition further comprises a fluorosurfactant.
 15. The aqueouscomposition of claim 14, wherein the organic iron-chelating reagent isselected from the group consisting of ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid;N,N,N′,N′-tetrakis(2-pyridylmethyl)ethane-1,2-diamine;1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid;N-(2-Hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid; and saltsthereof.
 16. The aqueous composition of claim 14, wherein the organiciron-chelating reagent comprises EGTA, wherein a molar ratio of ferriciron to the EGTA is about 0.04 to about 0.28.
 17. The aqueouscomposition of claim 14, further comprising2-hydroxypropane-1,2,3-tricarboxylate, wherein the organiciron-chelating reagent and the 2-hydroxypropane-1,2,3-tricarboxylate aredistinct molecules.
 18. The aqueous composition of claim 14, wherein thenon-ionic surfactant has a Hydrophilic-lipophilic balance of about 11 toabout
 16. 19. The aqueous composition of claim 14, wherein the non-ionicsurfactant is present at a concentration up to about 0.3% (mass/volume).20. The aqueous composition of claim 14, wherein the ferric iron isdissolved in the aqueous composition at a concentration of about 50 μMto about 350 μM.
 21. The aqueous composition of claim 14, furthercomprising polyvinylpyrrolidone.
 22. The composition of claim 14,further comprising an indicator dye.
 23. An isothermal nucleic acidamplification method, said method comprising: a) contacting thecomposition of claim 14 with a target sample to form an aqueous mixture;b) subjecting the aqueous mixture of step a) to a thermal lysis process;and c) subsequent to step b), subjecting a portion of the aqueousmixture to an isothermal nucleic acid amplification process.
 24. Themethod of claim 23, wherein isothermal nucleic acid amplificationprocess comprises loop-mediated isothermal amplification.
 25. The methodof claim 23, wherein the composition further comprises a bufferingagent, and an enhancer for facilitating a LAMP-BART reaction.
 26. Themethod of claim 23, wherein subjecting the mixture to thermal lysiscomprises heating the mixture to about 100° C. for about 15 minutes,wherein the cooled lysate is added to a control LAMP-BART pellet forisothermal amplification.